Project Summary We will characterize immune signatures (IMS) in individuals associated with four different states of Mycobacterium tuberculosis (MTB) exposure, namely those that have 1) a contained latent infection, 2) an uncontained infection characterized as active disease, 3) received BCG or experimental vaccination, or 4) unexposed individuals that were not vaccinated and with no evidence of past or present infection. We will recruit patients from multiple geographic sites (USA, Peru, Sri Lanka & Sweden), leveraging our established collaborations. The sites were selected so that for each exposure state we have a large number of donors from two separate locations available, thus ensuring generality of the defined IMS. IMS are the result of changes in the composition and activation / differentiation state of different cell populations. Our focus is on the contribution of MTB antigen-specific memory CD4 and CD8 T cells to shaping the IMS of the four exposure states. We will characterize the IMS of memory CD4 and CD8 T cells of individuals in all cohorts. In addition, we will isolate and characterize MTB antigen-specific CD4 and CD8 memory T cells using tetramer staining assays. This will identify particular CD4 and CD8 memory subsets in which MTB antigen-specific cells are located. For example, we have shown in latently infected individuals that memory CD4 T cells are nearly exclusively in the CCR6+CXCR3+CCR4? subset, and others have shown that CD8 memory T cells, and within these the CD8+ MAIT subset, are important in MTB infection. Consequently, we will isolate those CD4 and CD8 subsets that contain MTB antigen-specific cells by sorting them based on phenotypic markers and characterize their IMS. This will determine if the antigen-specific cells can be completely characterized based on their subset membership, or if their IMS shows additional differentiation. For a limited number of donors with latent infection and uninfected controls, we will have access to BAL samples that will allow comparison of T cell IMS in blood vs. lung. To obtain IMS we will perform unbiased transcriptomic (RNA-Seq) and proteomic (Mass spectrometry) analysis. We will further profile panels of markers that are known to impact T cell activation states and/or are identified as specifically up- or down- regulated in MTB antigen-specific cells based on data from our unbiased analysis. We will perform targeted profiling of phenotypic markers and cytokine production primarily by cytometry assays (FACS, CyTOF). These results will be further correlated with tertiary measures such as clinical outcomes, HLA type and other individual donor-related characteristics. Our results will be provided to Project 3 to profile the functionally relevant IMS utilizing epigenetic analyses, transcription factor Chip-Seq and RNAi.